Cell

ABSTRACT

The invention relates to the use of a cultured stem cell to produce a tooth progenitor cell.

FIELD OF THE INVENTION

[0001] The invention relates to the production of a tooth progenitorcell. In particular, the invention relates to the use of a cultured stemcell to produce a tooth progenitor cell.

BACKGROUND TO THE INVENTION

[0002] Teeth are essential organs for animal survival and of obviousclinical and/or cosmetic importance. There are many instances wheretooth replacement is desirable and current treatments are restricted toartificial prostheses or implants.

[0003] Tooth primordia explants can be cultured in vitro allowing avariety of manipulation studies including introduction of genes and/orproteins and tissue recombinations. Manipulated primordia can betransferred to renal capsules of adult animals (such as mice) to produceconditions for development of adult teeth. However, these culturetechniques require frequent animal sacrifice. This is one of theproblems associated with the prior art.

[0004] Prior art approaches to the production of tooth primordia reliedon in vitro tissue recombination. Two different tissue types wereindependently dissected from the animal embryo, and these wererecombined in the laboratory. Signals from one may then induce formationof tooth primordia in the other. This is a labour intensive processcarried out by highly trained workers involving a great deal of surgicalskill.

[0005] According to the prior art, the tissue requirements forprogression of tooth development change early in development. Forinitiation, it is thought that oral epithelium is essential and can formteeth when recombined with any mesenchymal cells, as long as they arederived from the neural crest. Thus, according to the prior art, neuralcrest derived cells are essential for the formation of tooth progenitorcells.

[0006] The present invention seeks to overcome at least some of theproblems associated with the prior art.

SUMMARY OF THE INVENTION

[0007] As explained above, production of tooth primordia has onlypreviously been accomplished using tissue recombination techniques. Itis surprisingly shown herein that production of tooth progenitor cellsmay be accomplished using cells cultured in the laboratory. Inparticular, tooth progenitor cells may be produced using embryonic stemcells (ES cells) cultured in the laboratory.

[0008] Accordingly, the present invention provides for the use of acultured cell to produce a tooth progenitor cell. Preferably, thecultured cell is a stem cell. More preferably, the cultured cell is anES cell.

[0009] The production of tooth progenitor cells from cultured stem cellsmay advantageously be accomplished by inducing said ES cells with oralepithelium.

[0010] A tooth progenitor cell is one which expresses certain molecularmarkers characteristic of tooth progenitor cells. For example, a cellwould be considered to be a tooth progenitor cell if it expressed one ormore tooth mesenchymal cell markers. Examples of such markers includeBarx1, Dlx2, Dlx5, Msx1, Pax9, Activin βA, Lhx6, Lhx7 and others. Thesemarkers may be detected by any suitable means, such as western blotting,immunofluorescence, radioactive in situ hybridisation or other suitablemeans, which are described in more detail below.

[0011] Oral epithelium may be from any suitable source, such as from amouse. The preparation of oral epithelium is discussed in more detailbelow.

DETAILED DESCRIPTION OF THE INVENTION

[0012] According to a first aspect, the invention relates to use of acultured cell to produce a tooth progenitor cell.

[0013] According to a second aspect, the invention relates to use of acultured cell to produce a tooth progenitor cell, wherein said culturedcell is a stem cell. The cultured stem cell may be a neural stem cell(NSC), or an embryonic stem cell (ES cell). Preferably, said culturedcell is an embryonic stem cell (ES cell).

[0014] According to a third aspect, the invention relates to a methodfor production of a tooth progenitor cell from a cultured cell, saidmethod comprising; providing a cultured cell, contacting the culturedcell with one or more oral epithelial cells, and incubating for a timesufficient to produce said tooth progenitor cell.

[0015] According to a fourth aspect, the invention relates to a methodfor production of a tooth progenitor cell from a cultured cell, whereinthe cultured cell is an ES cell, said method comprising; providing acultured ES cell, contacting the cultured ES cell with one or more oralepithelial cells, and incubating for a time sufficient to produce saidtooth progenitor cell.

[0016] According to a fifth aspect, the invention relates to a toothprogenitor cell produced from a cultured cell.

[0017] According to a sixth aspect, the invention relates to a toothprogenitor cell produced from a cultured cell wherein said cultured cellis an ES cell.

[0018] For ease of reference, these and further aspects of the presentinvention are now discussed under appropriate section headings. However,the teachings under each section are not necessarily limited to eachparticular section.

[0019] Preferable Features

[0020] Preferably, the cultured cell is a stem cell. More preferably,the cultured cell is an ES cell.

[0021] Incubation of the cultured ES cell with the epithelial cell(s) isfor a time sufficient to produce the tooth progenitor cell. Preferably,this time is about 72 hrs.

[0022] Preferably, tooth progenitor cell markers are as describedherein. Preferably, such markers are assessed by in situ hybridisation.

[0023] Advantages

[0024] The present invention has a number of advantages. Theseadvantages will be apparent in the following description.

[0025] By way of example, the present invention is advantageous since itrequires minimum animal sacrifice.

[0026] Further, the present invention is advantageous since it is laboursaving.

[0027] Further, the present invention is advantageous since it does notinvolve multiple surgical tissue recombination.

[0028] Tooth Development

[0029] Development of the mammalian tooth has been recognised as a modelsystem for study of epithelial/mesenchymal interactions duringorganogenesis. Teeth start to develop early in mammalian embryogenesis(11 days in mice, 6 weeks in humans), from a series of reciprocalinteractions between two cell types, oral epithelium and neuralcrest-derived mesenchyme cells. syndecan-1, Tgfβ-1, Tgfβ-3, Bmp-4,Bmp-7, Shh, CD44, Otlx-2, Lhx6, Lhx7, FGF8, Pitx2, or any other suitablemarker gene. This is discussed in more detail below.

[0030] Mesenchymal Markers

[0031] Examples of such markers include Barx1, Dlx2, Dlx5, Msx1, Pax9,Activin Aβ, Lhx6, Lhx7 and others. These markers may be detected by anysuitable means, such as western blotting, immunofluorescence,radioactive in situ hybridisation or other suitable means.

[0032] In wild type teeth at the bud stage Barx-1 gene expression isprincipally found in the molar region of the mandible and maxilla and ispresent in a broad field of neural crest derived mesenchymal cellsrather than being restricted to dental mesenchyme (Ferguson et al.,1998. Tissier-Seta et al., 1995).

[0033] Msx-1, Lef-1 and Bmp-4 are expressed in the dental mesenchyme(i.e. the condensing mesenchymal cells associated with invaginatingincisor and molar epithelial tooth buds) in response to epithelialsignalling (Ferguson et al., 1998: Mackenzie et al., 1991; Kratochwil etal., 1996; Vainio et al., 1993).

[0034] Dlx-2 expression is principally found in mesenchymal cellsimmediately surrounding the epithelial bud, but is also present in thedental epithelium on the buccal side of the buds (Ferguson et al., 1998;Thomas et al., 1995; Qui et al., 1997).

[0035] Pax-9, Lhx6 and Lhx7 are expressed in early tooth mesenchymeprior to bud formation and subsequently in condensing mesenchyme at thebud stage (Ferguson et al., 1998; Neubuïser et al., 1997).

[0036] Gli-3 is expressed in the mesenchyme from E10.5. At the bud andcap stage Gli-3 expression is slightly more localised than Pax-9expression, and is concentrated in the dental papilla and dentalfollicle (Ferguson et al., 1998; Hardcastle and Sharpe, 1998).

[0037] Inductive signals for tooth development come from the epitheliumwhereupon the responding mesenchymal cells are programmed to becomeodontogenic (2). Odontogenic mesenchymal cells then provide instructivesignals for further tooth development (3). The epithelial cellseventually give rise to ameloblasts which are responsible for enamelformation and mesenchyme cells form odontoblasts which produce dentine.

[0038] The identity of these different instructive signals has beenrevealed by gene expression studies and implantation experiments. FGF8,BMP4 and SHH are established as early instructive signals from the oralepithelium (3). BMP's, FGF's and activin are among the early signalsfrom the mesenchyme (3,4).

[0039] Key molecules involved in tooth organogenesis signalling includeActivin βA, which is expressed in presumptive tooth germ mesenchyme andis a signalling molecule in tooth development. Activin proteins areproduced from two gene products, Activin βA and Activin βB whichdimerise to form activin A (βA:βB), activin B (βB:βB) and activin AB(βA:βB). The closely related inhibins, inhibin A and inhibin B, aredimers consisting of an activin βA or βB subunit linked to aninhibin-specific α subunit (Vale et al., 1990; Roberts et al., 1991;Roberts and Barth, 1994).

[0040] Analysis of tooth development in Activin βA mutant embryos showsthat incisor and mandibular molar teeth fail to develop beyond the budstage. Activin βA is thus an essential component of tooth development,and is a mesenchymal cell marker gene.

[0041] Tooth Gene Expression Markers

[0042] Expression of well characterised mesenchymal and epithelialmarker genes may be examined by any suitable method. For example,radioactive in situ hybridisation may be performed on embryos, such asup to the bud stage. Suitable genes to examine the expression patternsof may include Barx-1, Msx-1, Dlx-2, Pax-9, Gli-3, Lef-1,

[0043] Syndecan-1, a cell surface heparin sulphate proteoglycan istransiently expressed in the dental mesenchyme and is thought toregulate dental mesenchymal cell condensation beneath the invaginatingdental epithelium (Ferguson et al., 1998; Thesleff et al., 1996).

[0044] Tgfβ-1 is found in the dental mesenchyme and weakly in theepithelium of the incisors and only appears in the molars in the dentalepithelium at the cap stage (Ferguson et al., 1998; Vaahtokari et al.,1991).

[0045] Tgfβ-3 expression is widespread in the mesenchyme of the face,but its expression appears to be substantially absent from thecondensing mesenchymal cells immediately adjacent to the epithelial budsof incisors and molars (Ferguson et al., 1998; Chai et al., 1994).

[0046] Epithelial Markers

[0047] Examples of such markers include Pitx2, p21, Wnt7b and others.These markers may be detected by any suitable means, such as westernblotting, immunofluorescence, radioactive in situ hybridisation or othersuitable means.

[0048] Genes known to be expressed in tooth germ epithelium includeBmp-7, Sonic hedgehog (Shh), CD44, FGF8, Pitx2 and Otlx-2 genes.

[0049] In wild-type embryos, Bmp-7 is initially expressed in the dentalepithelium, but expression shifts to the mesenchyme around the toothbuds from E13.5 (Åberg et al., 1997). At E13.5 mesenchymal Bmp-7expression is found only in lower incisors, which are the most advanceddevelopmentally at this stage, whereas expression persists in theepithelium of upper incisors and molars (Ferguson et al., 1998).

[0050] Shh is expressed in the epithelial thickening of early toothgerms and is thought to be an important component of the signals thatpass from the epithelium to the underlying mesenchyme at this earlystage, inducing gene expression in the mesenchyme and instructing it tobegin condensation (Bitgood and McMahon, 1995; Thesleff and Sharpe,1997). At later stages, Shh is down-regulated but transcripts reappearin the epithelial cells that constitute the enamel knot, a transientsignalling centre that arises in the dental epithelium at the late budstage of tooth development (Ferguson et al., 1998; Vaahtokari et al,1996).

[0051] CD44 and Otlx-2 are expressed more widely in the oral epitheliumthan Shh (Ferguson et al., 1998; Mucchielli et al, 1997). CD44 encodesthe hyaluronan receptor and Otlx-2 is the murine homologue of the humangene which when mutated, causes the disease known as Rieger syndrome inwhich teeth are absent (Semina et al; 1996).

[0052] Follistatin is an activin-binding protein that has been shown toinhibit the activity of activin (Michel et al., 1993; De Winter et al.,1996). The expression pattern of Follistatin may be examined by in situhybridisation analysis (Ferguson et al., 1998). Follistatin expressionis found in tooth germ epithelial cells immediately adjacent to activinβA expressing cells from E11.5. At later stages, follistatin transcriptsare restricted to the columnar-shaped cells that form the outermostlayer of the epithelial bud, while the central core of epithelial cellsare follistatin-negative (Ferguson et al., 1998). Follistatin istherefore expressed in the tooth epithelium adjacent to and in acomplementary pattern to activin βA in the tooth mesenchyme.

[0053] Tooth Primordia

[0054] Tooth primordia can be cultured in vitro allowing a variety ofmanipulation studies including introduction of genes and/or proteins andtissue recombinations. Most significantly, manipulated primordia can betransferred to renal capsules of adult animals (such as mice) to produceconditions for development of adult teeth. Advantageously, toothprogenitor cells according to the present invention may be used for theproduction of tooth primordia, or more fully developed teeth.

[0055] It is envisaged that a tissue engineering approach based on theteachings disclosed herein with respect to tooth development may be usedto generate teeth in vitro and ultimately in vivo in an adult oralcavity.

[0056] ES Cells

[0057] As shown herein, cultured cells such as ES cells can replaceneural crest-derived mesenchymal cells in production of tooth progenitorcells. Similarly, as disclosed herein. artificial epithelium may beengineered to emulate embryonic oral epithelium. It is envisaged thatthe cultured epithelial cells disclosed herein may be used in theproduction of artificial epithelium, and that this epithelium may beengineered to posess characteristics of oral epithelium, therebyallowing replacement of embryonic epithelium with engineered epitheliumin the production of tooth progenitor cells.

[0058] Other sources of stem cells that may replace neural crest-derivedmesenchymal cells may include primary adult neural stem cells. These maybe obtained using established methods, for example as described inJohansson et al (Cell vol. 96, p25 (1999)) and/or Clarke et al. (Sciencevol. 288, p1660 (2000)). Such cells are even capable of forming neuralcrest cells when transplanted into mouse blastocysts. Cultured neuralstem cell line(s) may also be used to replace neural crest-derivedmesenchymal cells according to the methods of the present invention.

[0059] It is envisaged that the tooth progenitor cells according to thepresent invention may be usefully employed to generate murine teethentirely from cultured cells, by combining ES cell aggregates withepithelium produced from immortalised cell lines and ES cells.

[0060] The ability of ES cells to form primitive ectoderm-like cellpopulations has been established (5,6). The combination of secretedsignals necessary to induce odontogenesis in such cells may be providedby experimental manipulation of the cells, for example using the beaddelivery system as described herein. Moreover, it is envisaged that theES cell-derived ectoderm may advantageously replace odontogenic ectodermonce the direction of signalling has transferred to the odontogenicmesenchyme.

[0061] Stem Cell Culture for Tooth Formation

[0062] As explained herein, the present invention involves method(s) forthe generation of a mesenchyme tissue capable of forming teeth fromcultured stem cells. The stem cells may be prepared for theinduction/interaction in a number of ways. For example, they may bepelleted to form small aggregates. This may be accomplished by pelletingthem onto filters. Such filters may comprise any suitable substrate,such as pregelatinised Millipore filters. For convenience, the filtersmay be supported by metal grids, for example as described in Ferguson etal. (1998). The stem cells may be pelleted into small holes made in agel or other suitable semi-solid support. The gel may be a collagen gel.The gel may be Collaborative Biomedical Products' Matrigel or a similarsubstrate. Epithelium is overlaid onto the stem cells to cover the holewhich is them covered with a thin layer of gel and incubated. Gels usedin this manner may themselves be supported by membrane(s) and/or metalgrids as outlined above and in the Examples section.

[0063] Tooth Replacement

[0064] The present invention may be used for tooth replacement,particularly for human tooth replacement. For example, this would beaccomplished using human ES cells in the methods of the currentinvention. It would be preferable to grow the tissue engineered teeth inthe adult oral cavity, thereby allowing direct tooth replacement.

[0065] It is known that embryonic tooth primordia can develop and grownormally in the adult environment, for example using renal transfers(1,4). It seems that sites such as the adult kidney and eye provide asuitable environment largely by allowing an adequate blood supply. Wetherefore envisage that tooth rudiments surgically implanted into theoral cavity should develop normally.

[0066] In addition to being able to develop a series of procedures toallow tooth replacement it is desirable that the tooth that develops insitu is of the correct shape and size. A number of the genes thatdetermine tooth shape are known, and by manipulation of these genes itis possible to change tooth shape (1,4,7,8). Similarly, it is shownexperimentally that modulation of signalling events leads to alterationof tooth size.

[0067] For example, inhibition of Wnt signalling leads to thedevelopment of smaller teeth (9). These observations could beadvantageously employed in the methods of the present invention.

[0068] The methods of the present invention could be usefully applied toa tissue engineering approach for the generation of a complete mammalianorgan, a tooth, de novo from cultured cells. The approach involves thereplacement of embryonic tissues that form teeth with tissues generatedfrom cultured cells as disclosed herein.

[0069] One aspect of the present invention concerns embryonic oralepithelium, that when recombined with mesenchyme derived from culturedembryonic stem cells (ES cells) induces tooth specific gene expressionand early tooth development in the ES cell tissue.

[0070] As is known in the art, the second branchial arch of mammalianembryos does not develop teeth. In one embodiment of the invention,using a double recombination technique with tissues derived fromgentically marked mouse strains, odontogenesis is induced in secondbranchial arch mesenchyme. It is further demonstrated that this tissueis then able to induce odontogenesis in second branchial archepithelium. Early stage tooth germs are then formed from theserecombined tissues according to the present invention. Formation of saidtooth germs is confirmed by monitoring expression of the appropriatemolecular markers as described above. Thus, it is an advantageousfeature of the present invention that non-odontogenic tissues can bemade to form teeth as disclosed herein.

[0071] In another embodiment, the invention relates to the induction ofES cell-based mesenchyme to undergo odontogenesis. Oral epithelium isrecombined with an embryonic stem cell mesenchyme. Epithelialinvaginations (tooth buds) are formed. Using molecular markers for toothbuds, it is demonstrated that expression of these markers is induced inthe ES cell mesenchyme around the epithelial invaginations, indicativeof early odontogenesis taking place and confirming tooth bud formation.

[0072] Thus, it is an advantageous feature of the present invention thatES cell based mesenchyme can be induced to undergo odontogenesis.

[0073] Advantageously, the methods of the present invention may beemployed to form teeth entirely from embryonic tissues that would notnormally form teeth.

[0074] It is envisaged that the present invention may enable ES cells toreplace neural crest-derived embryonic cells and permit toothdevelopment.

[0075] Advantageously, epithelial cells may be produced from cell linesand be used as a replacement for oral epithelium in vitro.

[0076] The methods of the present invention may be advantageouslyapplied to the replacement of the embryonic environment usually requiredfor tooth development by an adult environment, such as via implantationof tooth progenitor cells or structures resulting therefrom into anadult jaw for development. Teeth produced/transplanted according to thepresent invention continue to grow and develop when implanted into thejaw bone, and become attached thereto.

[0077] It is envisaged that the present invention provides for cells tobe directed to follow an odontogenic pathway in culture and subsequentlydevelop into mature teeth when implanted into mammalian kidney and/orjaw.

[0078] It is an advantage of the present invention that successful toothdevelopment can be produced from cultured cells.

[0079] It is demonstrated herein that odontogenic signals can be passedto mesenchymal and epithelial cells which normally do not form teeth byprogramming them in culture for subsequent tooth development accordingto the present invention.

[0080] It is an advantage of the present invention that mesenchymalcells can be programmed by exposure to odontogenic signals (eg. fromoral epithelium) in culture to subsequently form teeth on implantation(eg. renal implantation, or implantation into jaw).

[0081] It is an advantage of the present invention that tooth primordiaattach and develop successfully following implantation into the jaw.

[0082] It is envisaged that the present invention facilitates thereplacement of oral epithelium with cultured cell line(s), and/orfacilitates the replacement of epithelium with protein signals which maybe advantageously applied as protein and/or as gene(s) encoding same,and/or facilitates use of stem cells such as neural stem cells and/orembryonic stem cells for the production of tissue engineered teeth.

EXAMPLES

[0083] The present invention will now be described by way of example, inwhich reference is made to:

[0084]FIG. 1 which shows a stained section; and

[0085]FIG. 2 which shows a stained section.

[0086] In slightly more detail:

[0087]FIG. 1 shows a developing bud; and

[0088]FIG. 2 shows visualised expression of tooth progenitor markergenes.

[0089] General Methods

[0090] Experiments herein are carried out on murine embryo explantscultured using an established system (see for example Ferguson et al.,1998, et seq.). Recombinations are performed using established methodsinvolving separation of epithelium and mesenchyme with Dispase (4).Histology involves cutting and staining wax sections and in situhybridisation with gene markers utilises 35S RNA probes with differentprobes used on adjacent sections. Renal transfers are carried out in sixweek old male CD1 mice using a UK Home Office approved procedure, renalextracts are harvested over 10 to 16 days, decalcified in EDTA, serialsectioned and stained. Organ/tissue culture is according to methodsknown in the art, for example in (‘Organ culture in the analysis oftissue interactions.’ I.Thesleff and C.Sahlberg, from MolecularEmbryology Methods and Protocols Vol. 97. Ch.3, Ed.s PT Sharpe and IMason; Humana Press 1999). ES cells are propagated, maintained andprepared according to standard protocols, which may be found for examplein (‘CRE recombinase mediated alterations of the mouse genome usingembryonic stem cells.’ Hadjantonakis et al., from Molecular EmbryologyMethods and Protocols Vol. 97. Ch.8, Ed.s PT Sharpe and I Mason; HumanaPress 1999). Where appropriate, ES cells may be pelleted onto filters,or preferably are grown on pregelatinised filters using standard cultureconditions as explained above.

Example 1

[0091] Tooth Development from Non-odontogenic Embryonic Tissues

[0092] The extent to which teeth can develop from recombinations ofembryonic tissues that do not normally form teeth is determined.

[0093] Oral epithelium (first branchial arch) from E10 embryos isrecombined with mesenchyme from the second branchial arch. Theseexplants are cultured for three days to initiate odontogenesis in themesenchyme which is confirmed by in situ hybridisation with molecularmarkers.

[0094] Epithelial-mesenchymal Tissue Recombinations

[0095] Recombinations are carried out at E11.5 and E13.5. Molar anlagenof the mandibles are dissected out in DMEM with glutamax-1. Theepithelium and mesenchyme are isolated following incubation in asolution of Dispase (Gibco BRL) made up in calcium and magnesium freePBS at 2 units per ml for 10-15 minutes at 37° C. After incubation themandibles are washed in D-MEM with 10% foetal calf serum (FCS), and thetissues are mechanically separated using fine tungsten needles. Forrecombination, epithelium and mesenchyme are aligned in the correctorientation (as taught in Ferguson et al., 1998) on top of transparentNuclepore membrane filters (0.1 micron pore diameter; Costar). Therecombinations are cultured for 24 to 48 hrs in D-MEM with 10% foetalcalf serum, after which they are either fixed and processed forradioactive in situ hybridisation, or transplanted under the kidneycapsule of male adult mice and cultured for a further 10 days to allowfor full development of teeth.

[0096] In situ Hybridisation

[0097] Radioactive in situ hybridisation procedures are carried out onembryos at E10.5-E14.5 as described by Wilkinson (1995). The radioactiveantisense probes are generated from mouse cDNA clones such as activinβA, follistatin, Barx-1, Bmp-4, Bmp-7, CD44, Dlx-2, Fgf8, Gli-3, Otlx-2,Pax-9, Shh, Syndecan-1, Tgfβ-1, Tgfβ-3, or others as discussed herein.

[0098] After three days the oral epithelium is removed and replaced withsecond branchial arch epithelium. At this time odontogenic potentialresides in the second branchial arch mesenchyme (having been induced bythe oral epithelium) which then provides the inductive signals back tothe second branchial arch epithelium. Explants are cultured for afurther three days and assayed for formation of tooth buds by histologyand molecular markers.

[0099]FIG. 1 shows formation of tooth buds in this system (arrowed).

[0100] Identical explants are transferred to renal capsules todemonstrate that normal teeth can form from a combination of secondbranchial arch tissues that can not normally form teeth. It is importantin these experiments to ensure that there is no contamination of secondbranchial arch tissues with first arch cells. This is monitored by usingseparate embryos from ROSA 26 and GFP mice, whose cells may be easilydistinguished using the engineered markers therein. By harvesting firstarch tissues from GFP mice and second arch tissues from ROSA 26 mice theorigins of all cells in the teeth formed can be assayed by Lac Zstaining and GFP detection.

[0101] Induction of Tooth Primordia from Mesenchymal Tissue

[0102] In vitro induction of tooth primordia from mesenchymal tissue isdemonstrated. The the necessary signals are provided artificially usingprotein soaked beads. These tooth progenitors are then allowed todevelop into fully grown teeth in vivo in adult animals.

[0103] Mandibles from embryos at E11.5, E12.5 and E13.5 are dissected inD-MEM with glutmax-1 (Gibco BRL). The rest of the embryo is used forgenotyping. For cultures at E11.5 and E13.5, individual molar toothanlagen are isolated from surrounding oral and aboral tissue, while forcultures at E12.5, whole mandibles are used. These are placed with oralsurfaces facing upwards, on membrane filters supported by metal gridsfollowing the Trowell technique as modified by Saxen (Trowell, 1959;Saxen, 1966). Affi-Gel agarose beads (BioRad) are washed several timesin PBS then are dried out before being added to recombinant activin Aprotein at 1 mg/ml, a concentration known to be able to induce mesodermformation in Xenopus animal cap assays (Ferguson et al., 1998), or BSAat the same concentration for one hour at 37° C. The beads are pushedinto the mesenchyme so that they lay in close proximity to thedeveloping tooth germs. The mandible explants at E11.5 and E13.5 arecultured with beads for 24 to 72 hrs in D-MEM with 10% foetal calfserum. The E12.5 mandible explants are cultured with beads for 6 days,then fixed in 4% paraformaldehyde (SIGMA) and processed for histologicalexamination using haemotoxylin/eosin staining. A standard incubator isused at 37° C. with an atmosphere of 5% CO₂ in air and 100% humidity.All solutions contain penicillin and streptomycin at 20 IU/ml. After theperiod of culture, the E11.5 and E13.5 explants are removed from theirmembrane filters and transplanted under the kidney capsules of maleadult mice. During this procedure, most of the beads are dissociatedfrom the explants. The explants are cultured in host kidneys for 10 daysto allow for full development of teeth. The resulting tissues are thenfixed in Bouin's solution (SIGMA), dehydrated, and embedded. Serialsections of 7 microns are cut and stained using alcian blue/chlorontinefast red.

[0104] Induction of Tooth Progenitor Cells Using Artificial Fgf-8/Bmp-4Signals

[0105] Mandibles are dissected at E11.5. Where indicated, epithelium isremoved after incubation in Dispase (2 units per ml) for 10 minutes at37° C. For the application of Fgf8 protein, heparin acrylic beads(Sigma) are washed and then incubated overnight in 1 mg/ml Fgf-8 protein(recombinant mouse FGF-8b; R&D Systems, Europe) at 4° C. For theapplication of Bmp-4 protein, Affi-Gel agarose beads are washed andsoaked in the protein (recombinant human BMP-4) for 1 hr at 37° C. Aconcentration of 100 μg/μl Bmp-4 was used, since this concentration hasbeen shown to inhibit Pax-9 expression in a similar assay (Neubüser etal, 1997). After 24-48 hrs in culture the explants are fixed andprocessed for in situ hybridisation. Digoxygenin wholemount in situhybridisation is carried out as described by Pownall et al (1996).

Example 2

[0106] Use of Cultured Cells in the Production of Tooth Progenitor Cells

[0107] Replacement of neural crest-derived mesenchymal cells with EScells, and subsequent determination of these as tooth progenitor cellsis demonstrated.

[0108] Disclosed herein is a way of reproducibly producing a solidmesenchymal tissue from cultured cells such as ES cells, said tissuebeing capable of interacting with oral epithelium and forming teeth.

[0109] In order to establish that ES cells can be used to replace neuralcrest-derived mesenchymal cells, mouse ES cells are pelleted ontofilters to form small aggregates. These aggregates are overlaid withoral epithelium from E10 mouse embryos and allowed to develop for threedays in culture. Histology of the cultures reveals evidence ofepithelial invaginations with surrounding expression of the toothmesenchymal marker genes Barx1 and Dlx5 as shown in FIG. 2.

[0110] Wild type 129 ES cells are grown and maintained in a pluripotentstate using the same conditions as are routinely used for genetargeting. Cells are pelleted onto pre-gelatinised Millipore filters andallowed to grow for 1 to 3 days to form aggregates. ES cell aggregatesare overlaid with E10 oral epithelium from ROSA 26 embryos and culturedfor three to five days. Histology, molecular markers and renal transferanalysis are carried out as described herein. As a control, ES cellshomozygous for a targeted allele of Msx1 will be used. Msx1 +/− ES and−/− cells are produced by re-electroporation of +/− ES cells with theoriginal targeting construct followed by selection with a higherconcentration of G418. Since Msx1 −/− embryos do not develop teeth, −/−ES cells do not permit tooth development beyond the bud stage whenrecombined with oral epithelium (10).

[0111] Furthermore, the protocol outlined in Example 1 above may befollowed where, after induction of odontogenic potential in the ES celltissue, the oral epithelium is replaced by non-odontogenic secondbranchial arch epithelium.

Example 3

[0112] Use of Cultured Cells in the Formation of Oral Epithelium.

[0113] The replacement of oral epithelium with odontogenic epithelialcultured cell lines is demonstrated.

[0114] It is taught herein how to replace oral epithelium with anepithelium derived from immortalised lines of tooth epithelial cells.Firstly, a number of clonal lines of immortalised cells derived fromearly odontogenic epithelium are generated.

[0115] Epithelial Cultures

[0116] Oral epithelium is isolated after incubation of E11.5 mandiblesin Dispase (2 units per ml) for 10 minutes at 37° C. The epithelium iscultured in Matrigel (Collaborative Biomedical Products) on membranefilters supported by metal grids as described in (Ferguson et al.,1998). Matrigel is a solubilised basement membrane extracted from theEngelbreth-HolmSwarm mouse sarcoma cell line and provides a matrixwithin which epithelial cells can develop. The gel sets rapidly andirreversibly at temperatures between 22° C. to 35° C. Therefore, theproduct is kept on ice and pre-cooled pipettes used. The filters arecovered with Matrigel which is left to set at 37° C. before theepithelia are pipetted on top. To visualise the epithelia they areweakly dyed with neutral red before being placed onto the Matrigel.Affi-Gel agarose beads (BioRad) soaked in recombinant activin A protein(1 mg/ml) or BSA are prepared as described herein. Beads are placed ontop of the epithelia, which are then topped with more Matrigel so thatthe cultures are surrounded. The epithelia with beads are cultured for48 hrs in D-MEM with 10% foetal calf serum. After the period of culture,cultures are washed in ice-cold methanol for 1 minute and then fixed infresh 4% paraformaldehyde for 1 hour at RT. Cultures are then preparedfor ³⁵S section in situ hybridisation.

[0117] Immortalised lines of odontogenic epithelial cells are produced.The cell lines generated are tested for their odontogenic inductivecapacities. Lines are established that express amelogenins, unique toothspecific proteins involved in enamel formation. Molecular markers areanalysed to determine whether the signalling properties of early oralepithelium are well established.

[0118] Cells that have properties of ameloblasts are identified byscreening the lines for amelogenin expression. RTPCR is used to screenfor expression of FGF8, BMP4, SHH and Pitx2 (the earliest marker of oralepithelium) to determine which lines are likely to be able to replaceoral ectoderm.

[0119] To test the odontogenic inducing capacity of cell lines,mesenchymal explants (epithelium removed) from mandibular primordia ofE12 embryos are cultured on filters in small wells in collagen gels.Cultured cells from the screened lines are layered onto the mesenchymalexplants and the explants cultured for 3 days. The extent of epitheliumformation and invagination is assayed histologically and with toothmolecular markers.

[0120] Since at E11.5 odontogenic inducing capacity resides in themesenchyme, naïve epithelium responds to these signals and allows toothdevelopment. If the growth medium used in the cultures does not containthe factors required for the cells lines to produce an odontogenicepithelium, conditioned media from cultures of intact mandibularprimordia explants are used.

[0121] The immortalised cell lines' inductive odontogenic properties areassayed by following the methods of the above examples, but replacingmandibular primordia mesenchyme with second branchial arch mesenchyme.

[0122] If the epithelial cells do not properly induce odontogenesis, theexpression of inductive signalling molecules (FGF8, BMP4, SHH etc.) isassayed in the collagen explant cultures and any missing signals arereplaced either by purified proteins on beads or by electroporation ofgene expression constructs (see below).

Example 4

[0123] Engineering Artificial Epithelium

[0124] As disclosed herein, oral epithelial inductive signals may besupplied using purified proteins. The following experimental approachesare used to reproduce (replace) inductive signals in oral epitheliumproduced from immortalised cultured cells/ES cells.

[0125] A number of signalling proteins have been identified that aresecreted by oral epithelium that direct mesenchymal cells to becomeodontogenic and generate epithelial tooth buds. These signals includeFGF8, BMP4 and SHH (3,11). These factors may be sufficient to inducetooth initiation in non-odontogenic cells.

[0126] The feasibility of engineering an artificial oral epithelium istested. Beads soaked in a combination of signalling proteins involved ininitiation are implanted into the epithelium of second branchial archextracts and tooth development analysed.

[0127] Teeth do not develop on the second branchial arch and so anyevidence of tooth development following addition of exogenous signalsimplicates initiation. A range of concentrations and combinations aretested.

[0128] Structures are formed in the explants treated with factors thatare not formed in control explants.

[0129] Alternatively, electroporation may be used to transfer geneexpression constructs into localised epithelial sites to reproduce thefirst brachial arch spatial arrangements of these three signallingmolecules in the second branchial arch.

Example 5

[0130] Production of ES Cell Derived Epithelium

[0131] In the production of tooth progenitor cells according to thepresent invention, it is desirable that oral epithelium be replaced byan ES cell-derived (ie. cultured cell-derived) epithelium.

[0132] The generation of animal tooth progenitors such as human toothprogenitors will preferably be accomplished entirely from culturedcells. A convenient source of human cells is cultured ES cells. Theability of ES cells to replace oral epithelium is demonstrated.

[0133] ES cells have been shown to be capable of formingectoderm/epithelial cells and following similar lines to those describedabove for immortalised epithelial cell lines, the requirements for EScells to generate an inductive odontogenic epithelium are investigated.

[0134] For control ES cells Pitx2 −/− ES cells are used since Pitx2 isexpressed in early oral epithelium and Pitx2 −/− embryos fail to formcap stage tooth buds (12,13,14).

[0135] Preferably, both odontogenic epithelium and mesenchyme arereplaced with ES generated epithelium and mesenchyme in a single explantin the methods of the above examples.

Example 6

[0136] Development of Tooth Explants in the Oral Cavity

[0137] Tooth explants can develop normally in at least two adult sites,the renal capsule and anterior chamber of the eye. It is determinedwhether explants may develop in the adult oral cavity.

[0138] Adult male mice are anaesthetised and single first molar teethextracted. Molar tooth primordia explants cultured for 3 days as in theabove examples are transplanted into the extraction site. The oralmucosa are closed over the explant, sutured and left for 10-16 days todevelop during which time the animal is fed a soft diet.

[0139] The extent of explanted tooth development is assayed byhistology. These experiments require the use of microsurgery. Surgicalexpertise is first acquired on sacrificed mice before being carried outon live animals.

[0140] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

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1. Use of a cultured cell to produce a tooth progenitor cell.
 2. Use ofa cultured cell to produce a tooth progenitor cell, wherein saidcultured cell is a stem cell.
 3. Use of a cultured cell to produce atooth progenitor cell, wherein said cultured cell is an embryonic stemcell (ES cell).
 4. A method for production of a tooth progenitor cellfrom a cultured cell, said method comprising; i) providing a culturedcell, ii) contacting the cultured cell of (i) with one or more oralepithelial cells, and iii) incubating for a time sufficient to producesaid tooth progenitor cell.
 5. A method according to claim 4 wherein thecultured cell is an ES cell.
 6. A tooth progenitor cell produced from acultured cell.
 7. A tooth progenitor cell produced from a cultured cellwherein said cultured cell is an ES cell.